Summary: Embryonic stem (ES) cells have the potential to differentiate into progenitor cells that are committed to a given lineage. There has been an explosion of interest in using ES cells, or their committed progenitor offspring, to repair or replace damaged tissues, yet the immune response to such cells across histocompatibility barriers has been poorly defined. This project examines the immmune response to ES cells and their committed progenitor offpring, and to genetically engineer ES cells to inhibit graft rejection. Early studies have revealed that uncommitted ES cells express no classical MHC class I molecules and small but varying amounts of the non-classical class I MHC CD1d. Such cells elicit weak CTL responses across syngeneic or allogeneic barriers and form tumors across syngeneic barriers. We have found that ES cells are killed by NK and LAK cells with lysis highly dependent on LFA-1/ICAM interactions. We found a similar response to neural progenitor cells (NP)which are similar to their ES founders in failing to express MHC molecules and expressing high levels of ICAM. In contrast, embryonal carcinoma cells (EC), which arise in syngeneic animals injected with ES cell populations are not killed by NK or LAK cells. The failure to kill EC cells appears to relate in some EC cells to lack of expression of ICAM and in others, an increased expression of both classical and non-classical class I MHC molecules. The mechanism by which EC cells develop from ES cells is also being explored. Because of the tumorigenic potential of ES cells, prevention of rejection cannot be aimed at ES cells, but will instead be aimed at non-tumorigenic committed progentior or mature differentiated cells. To protect such committed or differentiated cells from rejection, our strategy is to clone protective molecules, such as Fas-ligand, into ES cells and place them under control of a lineage specific promoter, so that they will not be expressed until the cell assumes a differentiated (non-malignant) phenotype. Additionally, we may clone into an ES cell line a suicide gene expressing an enzyme under control of an ES cell specific promoter.